Mycobacterium tuberculosis infects one-third of the world?s population and causes 2 million deaths annually. The rate of drug resistance in this pathogen is increasing globally, and many multidrug-resistant strains now have emerged that are difficult to treat. M. tuberculosis can cause both pulmonary and extraplumonary infections, but the pathogen and host factors that contribute to disease specificity and severity are not known. One of the common themes in bacterial pathogenesis that has emerged over the last decade from analysis of the molecular population genetics of many pathogenic bacteria is that the clone or cell line is the unit of virulence. This observation indicates that there is inter-clonal variance in relative virulence. Moreover, there are strongly nonrandom associations of bacterial clones and overall character of host-pathogen interactions. For example, it is common in species of pathogenic bacteria to have disease specialist clones and host specialist clones. These nonrandom associations usually are not revealed until overall genomic relationships among isolates of the species are resolved by molecular population genetic strategies. Analysis of this type also requires the collection of detailed patient data, which then can be linked to the genetic data available for the infecting bacterial strain. We have initiated a large collaborative study to analyze the molecular population genetics of M. tuberculosis virulence. As a first step in this process, we have developed a synonymous single nucleotide polymorphism (sSNP)-based population genetic framework for the study of M. tuberculosis isolates recovered from patients with defined clinical syndromes. This strategy is done in a high-throughput format, semi-automated format and can rapidly assign strains to particular clonal lineages on the basis of sSNP profile. We have conducted an initial analysis of >400 M. tuberculosis strains for >200 sSNPs and developed the sSNP genotyping scheme in a proof-of-principle study. The sSNP genotyping system will also be extremely useful for rapid molecular typing of strains for molecular epidemiologic studies, and tracing of strains for public health purposes, including multidrug-resistant organisms.